Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα(11)), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca(2+)(i)) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα(11) mutations mediate uveal melanoma development by constitutively up-regulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα(11) protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca(2+)(i) alterations associated with FHH2- and ADH2-causing Gα(11) mutations, and evaluated the influence of germline gain-of-function Gα(11) mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα(11) mutant. WT and mutant Gα(11) proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca(2+)(i) and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca(2+) (Ca(2+)(o)) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca(2+)(i) responses of FHH2- and ADH2-associated Gα(11) loss- and gain-of-function mutations, respectively. ADH2-causing Gα(11) mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca(2+)(o) stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα(11) mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα(11) mutations induce non-constitutive alterations in MAPK signaling.